The objective of this research project is to investigate the possibility that folate deficiency, which is a common human nutritional state, may play an important role in carcinogenesis by promoting genetic instability. Folic acid deficiency is associated with numerous chromosomal changes and enhances the genetic damage caused by mutagens. Therefore, folic acid supplementation may be a cancer control strategy relevant to large populations to reduce the risk of cancer in people exposed to mutagens/carcinogens. The first specific aim is to determine whether the combination of alkylating agents and nutritional folate deficiency causes an increased frequency of intragenic deletions in human stem cells and lymphoblasts. CD34+ human stem cells obtained from umbilical cord blood will be cultured with growth factors in folate replete or deficient medium. In other experiments, human lymphoblast cell lines will be grown in folate replete or deficient medium. Cells will be treated with ethyl methanesulfonate or hydroperoxycyclophosphamide. Mutant frequencies at the hprt (hypoxanthine guanine phosphoribosyltransferase) locus will be determined, and multiplex PCR and RT-PCR DNA sequence analyses peformed. The goal of this aim is to determine if folate supplementation can reduce the risk of deletions caused by alkylating agents in human cells. The second specific aim is to characterize the breakpoints in deletions resulting from the combination of folate deficiency and alkylating agents. CHO cells, CD34+ stem cells, and human lymphoblasts known to have intragenic deletions at the hprt locus after alkylating agent treatment in high and low folate states will be studied. The breakpoints will be sequenced after long PCR and rapid amplification of cDNA ends (RACE) and analyzed for common features. These studies will provide insights into the mechanisms by which the deletions were created. The third specific aim is to investigate the interaction of nutritional folate status and chemotherapeutic drugs on mitochondrial DNA damage. Liver samples collected from rats with deficient, replete or high folate status and treated with cyclophosphamide, doxorubicin, 5-fluorouracil or saline will be studied. Mitochondrial DNA will be isolated and analyzed for deletions and amplifications using short PCR and long PCR. High frequency deletions will be confirmed by DNA sequencing performed across the breakpoints. Some cancers have mitochondrial genome alterations including deletions and amplifications; these studies will determine if folate deficiency affects the creation of these types of events in the mitochondria. These experiments will indicate if correction of folate deficiency decreases the frequency of DNA damage caused by alkylating agents, thus supporting the concept that folic acid may be an effective chemopreventive agent, especially for second malignancies induced by alkylating agents.
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